This proposal deals with the function of nuclear protein kinases in the regulation of gene activity. It is concerned with a characterization of nuclear protein kinases with a special emphasis on their protein substrate specificity and an examination of the hypothesis that RNA synthesis is regulated by a direct phosphorylation of RNA polymerase. The activity of highly purified Novikoff ascites tumor RNA polymerase I and II is stimulated by the presence of a purified nuclear protein kinase. Experiments will be performed to establish the RNA polymerase subunit(s) phosphorylated in vitro and also to establish whether or not a causal relationship exists between protein phosphorylation and the stimulation of polymerase activity. The in vivo pattern of phosphorylation of RNA polymerase I and II will also be determined. The effect of phosphorylation on the selectivity of transcription by RNA polymerase II will be determined using SV40 and adenovirus DNA templates. The substrate specificity of the two major protein kinases present in nuclei of ascites tumor cells will be determined by phosphorylating isolated NHC proteins, as well as NHC proteins associated with DNA in a chromatin template, and resolution of the labeled proteins by two-dimensional gel electrophoresis. The in vivo pattern of NHC protein phosphorylation will also be determined in order to evaluate potential significance of specific protein kinases in the regulation of gene activity.